Tuberculosis (TB) is a leading cause of mortality from an infectious disease in the world, second only to HIV/AIDS. The WHO reports that in 2013 there were 1.5M deaths from TB. When a case of TB does not respond to two or more of the first-line drugs available, the infection is considered to be multidrug-resistant (MDR-TB). The rising incidence of MDR-TB is leading to poor patient outcomes since the second line drugs are introduced at a late stage of the disease. These drugs are expensive, require injection and have serious side effects, therefore cannot be administered before a diagnosis of MDR-TB occurs. The majority of TB cases are seen at peripheral health clinics, with no access to current diagnosis technologies. Early diagnosis, before the failure of the first line drugs, is needed for effective treatment, as well as for isolation of infected individuals. We propose to develop a test that will detect the 15 known Mycobacteria tuberculosis alleles that are found in 96-98% of resistance to the main first line drugs, Rifampicin and Isoniazid, using our hand-held, battery powered TangenDx Point of Care (POC) diagnosis system. TangenDx consists of a sample preparation device, which is followed by mycobacterial capture via filtration, at which point, lysing and amplification solutions are introduced. Lysis is performed by sonication, after which the sample is introduced into a disposable assay disk for DNA extraction and amplification, using isothermal amplification and real time detection via fluorescent intercalating dyes. The assay amplifies the hspx gene, which is unique to MTB. A built-in precise temperature controller enables the use of the highly robust and versatile loop-mediated isothermal amplification (LAMP). STEM primers are used with the LAMP technology to accelerate reactions. We were able to detect 8-40 CFUs of TB per disk assay or 20 genomic copies with >95% confidence. We propose to add DST to our TangenDx TB Assay to enable detection of 96-98% of all known MDR-TB strains by following these specific aims: In Specific Aim 1, LAMP primer sets specific to the three genetic locations associated with resistance to rifampicin and isoniazid will be designed and screened. STEM primers to each of the mutant alleles will be designed and tested for their ability to selectively accelerate LAMP reactions using synthetic DNA targets. Finally, we will determine the conditions that allow amplification and multiplexing of the three MDR-TB genetic regions (rpoB, katG and inhA) in the central chamber of the Tangen Dx Disk prior to STEM specific acceleration of specific LAMP reactions in the detection chambers. In Specific Aim 2, the optimized primer sets and conditions of Aim 1 will be tested with the whole TangenDx TB Assay system using characterized drug resistant TB strains spiked into healthy human sputum samples. The proposed system would make quick MDR-TB diagnosis accessible and feasible in regions of the world where such technologies are lacking, and where TB is most widely spread. A timely diagnosis, with an accurate DST evaluation, could significantly lower TB associated mortality and costs of care worldwide.